The in vitro excystation of Sarcocystis gigantea sporocysts

Studies on the in vitro excystation of Sarcocystis gigantea sporocysts revealed that pretreatment before exposure to trypsin and bile was an essential prerequisite. However, in contrast to Sarcocystis tenella and Sarcocystis capracanis, incubation in cysteine hydrochloride under CO2 was largely unsuccessful for excysting Sarcocystis gigantea: of the pretreatments tested, only exposure to sodium hypochlorite proved effective. Excystation from sodium hypochlorite-pretreated S. gigantea sporocysts took place in trypsin and bile between temperatures of 30 and 43 degrees C and occurred rapidly at 39 degrees C. While the presence of bile or bile salts was essential for this process, that of trypsin was not, although more sporocysts excysted in its presence than in its absence. Excystation occurred in the presence of all bile types tested but not when Tween 80 was substituted for bile. The highest levels of excystation were recorded when cattle or sheep bile or sodium taurocholate were used and the lowest when chicken or pig bile were employed. Neither the concentration of sheep bile above 2.5%, nor hydrogen ion concentration (pH range 5.0-10.0) appeared to have any marked effect on the level of excystation obtained.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

The survival of Sarcocystis gigantea sporocysts following exposure to various chemical and physical agents.

Using in vitro excystation as a measure of viability, it was found that at 4 degrees C Sarcocystis gigantea sporocysts survived considerably better in tap water (85% excystation after 174 days) than in either 2.5% potassium dichromate (15% excystation after 174 days) or 2% sulphuric acid (0% excystation after 5 days). Although they were able to resist 48 h suspension at room temperature in most laboratory reagents and disinfectants tested, six (sulphuric acid, ammonia, methanol, ethanol, potassium hydroxide, sodium hydroxide, Medol) had substantial sporocysticidal properties. Further investigation with three of these showed that sporocyst excystation was reduced from 65% to less than 10% following contact with 2.5% sulphuric acid for 1 h or with 2% ammonia or 4% Medol for 4 h. Sporocysts were either killed or had their ability to excyst severely impaired by heating to 60 degrees C and 55 degrees C for 5 and 60 min, respectively, by exposure to ultraviolet radiation at a dose of 4000 ET, or by prolonged storage in water at 24 degrees C. Sporocysts exposed to either constant or intermittent freezing at -18 degrees C suffered a comparatively slow decline in excystation rate with time, as did those subjected to desiccation. The duration of survival of desiccated sporocysts was inversely related to relative humidity and after 245 days at 33% relative humidity and temperatures of 15 degrees C or 24 degrees C, 60% of such sporocysts excysted.     

Sarcocystis Infection in Sheep from South-Western Norway

The occurrence of Sarcocystis infection and pathological changes were recorded in samples of the heart, diaphragm, and oesophagus from 198 healthy sheep representing 3 different age groups, obtained from an abattoir.The infection rate of S. gigantea (syn. S. tenella) was 18.2 %, and the distribution within groups was: ewes 30.0 %, yearlings 11.6 %, lambs nil. The infection rate of S. tenella (syn. S. ovicanis) was 65.1 %, and the corresponding distribution was: ewes 83.5 %, yearlings 74.4 %, and lambs 25.0 %. A third type of Sarcocystis sp. displaying thick wall was found in 3 samples.Focal interstitial infiltrates of mononuclear cells were demonstrated in 47.9 % of the hearts, in 19.6 % of the diaphragms and in 31.3 % of the oesophagi. The occurrence of Sarcocystis and the focal interstitial mononuclear cell infiltrates were positively correlated (P < 0.0001). Morphologically identical sporocysts typical of S. tenella were produced by dogs and foxes fed naturally infected sheep tissues. A cat fed S. gigantea macrocysts produced sporocysts characteristic for the species.Sarcocystis; pathology; life cycle; final hosts; sheep.     

In vitro and in vivo interaction between sporocysts of Sarcocystis muris and mouse peritoneal macrophages

The interaction between the sporocysts of Sarcocystis muris and mouse peritoneal macrophages was studied both in vitro and in vivo in an attempt to determine whether or not resident peritoneal macrophages might effect the excystation of S. muris sporozoites from sporocysts injected intraperitoneally. Sporocysts of S. muris were phagocytosed by peritoneal macrophages both in vitro and in vivo. The addition of either unheated mouse serum or fetal calf serum did not significantly alter the level of phagocytosis. The percentage of phagocytosis in vivo and by thioglycolate-, proteose peptone- and BCG-elicited macrophages in vitro was greater than that shown by unstimulated macrophages in vitro. After 8 h incubation in vivo and in vitro a small proportion of sporocysts (less than 5%) was seen to have collapsed walls and up to 5% to have stained sporozoites, suggesting increased permeability of the sporocyst wall. The significance of increased permeability of the cyst wall in the process of sporozoite excystation is discussed.     

Moxidectin and ivermectin metabolic stability in sheep ruminal and abomasal contents

The oral administration of macrocyclic lactones to sheep leads to poorer efficacy and shorter persistence of the antiparasitic activity compared to the subcutaneous treatment. Gastrointestinal biotransformation occurring after oral treatment to ruminant species has been considered as a possible cause of the differences observed between routes of administration. The current work was addressed to evaluate on a comparative basis the in vitro metabolism of moxidectin (MXD) and ivermectin (IVM) in sheep ruminal and abomasal contents. Both compounds were incubated under anaerobic conditions during 2, 6 and 24 h in ruminal and abomasal contents collected from untreated adult sheep. Drug concentrations were measured by high-performance liquid chromatography with fluorescence detection after sample clean up and solid phase extraction. Neither MXD nor IVM suffered metabolic conversion and/or chemical degradation after 24-h incubation in ruminal and abomasal contents collected from adult sheep. Unchanged MXD and IVM parent compounds represented between 95.5 and 100% of the total drug recovered in the ruminal and abomasal incubation mixtures compared with those measured in inactive control incubations. The partition of both molecules between the solid and fluid phases of both sheep digestive contents was assessed. MXD and IVM were extensively bound (>90%) to the solid material of both ruminal and abomasal contents collected from sheep fed on lucerne hay. The results reported here confirm the extensive degree of association to the solid digestive material and demonstrates a high chemical stability without evident metabolism and/or degradation for both MXD and IVM in ruminal and abomasal contents.     

Prior or concurrent exposure to different species of avian Eimeria: effect on sporozoite invasion and chick growth performance.

The effects of prior (immunity) or concurrent administration of Eimeria acervulina or Eimeria tenella on cellular invasion in vivo and in vitro and on growth performance in white leghorn chickens (WLC) were examined. Weight gains of WLC immunized with E. acervulina and challenged with E. tenella were significantly greater than those of nonimmunized chicks challenged with E. tenella (this occurred despite the increased invasion by E. tenella in E. acervulina-immunized chicks that was reported earlier). The weight gains and modest but consistent improvements in intestinal lesion scores, feed conversion ratios, and oocyst shedding in immunized/challenged WLC indicated that E. acervulina conferred a small measure of protection against E. tenella infection that was independent of the effect on invasion. In contrast, immunization of WLC with E. tenella significantly decreased (41%-51%) invasion by E. acervulina as compared with that in nonimmunized WLC but had little effect on chick growth performance. Concurrent inoculation of chicks with E. tenella and E. acervulina had little effect on invasion by E. tenella sporozoites or on subsequent performance of the chicks. In vitro, prior exposure of cultured cells to either of two isolates of E. tenella also caused a significant decrease in invasion by E. acervulina. No gross changes occurred in the culture morphology between the E. tenella-inoculated and noninoculated cultures. Collectively, the data indicate that prior exposure of WLC and cultured cells to single isolates of avian coccidia markedly influenced invasion by other species but had less effect on the growth performance of the birds.     

Experimentally induced monensin-resistant Eimeria tenella and membrane fluidity of sporozoites

Two resistant lines of Eimeria tenella (H) to monensin were developed after 35 passages in chickens medicated with 100-125 ppm or 125-200 ppm monensin in the diet. Drug sensitivity of the induced lines to different level drugs were estimated with mean lesion scores (LS), mean oocyst productions (OP), percentage optimum anticoccidial activity (POAA), reduction of lesion scores (RLS), relative oocyst production (ROP), anticoccidial index (ACI) and global index (GI), respectively. Membrane fluidity of sporozoites of the sensitive line (i.e. the parent line, coded as MON-S((S))) and two resistant lines (coded as MON-R((S))-1 and MON-R((S))-2) with and without in vitro exposure to monensin were determined. Membrane fluidity of MON-R((S))-1 and MON-R((S))-2 were significantly lower than that of MON-S((S)). In vitro exposure to monensin significantly increased membrane fluidity of MON-S((S)), but had a much less effect on those of MON-R((S))-1 and MON-R((S))-2. Sporozoits of the MON-S((S))and MON-R((S))-2 with or without in vitro exposure to monensin were examined by SEM, and the sensitive sporozoites (MON-S((S))) appeared swollen and bulgy after treatment with monensin, while there was no obvious morphological deformation in the resistant sporozoites (MON-R((S))-2). The results suggest that the altered membrane fluidity in the membranes of E. tenella may be related to the decreased sensitivity to monensin.     

Factors influencing enhanced Salmonella typhimurium infection in Eimeria tenella-infected chickens

To investigate a possible mechanism involved in the enhancement of Salmonella typhimurium infection in chickens concurrently infected with Eimeria tenella, S typhimurium was given orally to chickens 7 days after E tenella inoculation. The number of viable S typhimurium decreased in the ceca of chickens not inoculated with E tenella, whereas the number gradually increased in the ceca of chickens inoculated with E tenella. Cecal contents were analyzed for pH value, oxidation-reduction potential, and amounts of short-chain fatty acids and bile acids. In the ceca of E tenella-inoculated chickens, the oxidation-reduction potential significantly (P less than 0.05) shifted to the oxidative phase, and the concentration of volatile fatty acids (acetic acid, propionic acid, and butyric acid) significantly (P less than 0.05) decreased. In both aerobic and anaerobic incubations, the number of viable S typhimurium in vitro decreased as the molar concentration of fatty acids increased. Experimental evidence indicated that multiplication of S typhimurium in the ceca of E tenella-inoculated chickens was associated with decreased concentrations of volatile fatty acids.     

Differences in plasma and abomasal kinetics of albendazole and its metabolites in calves grazed on pasture or fed a grain-based diet

We evaluated the comparative plasma and abomasal fluid disposition kinetics of albendazole (ABZ) and its metabolites in calves either grazing on pasture or fed a grain-based concentrate diet. Six male Holstein calves (weight 180 to 200 kg) were allowed to graze on lush pasture for three weeks before intraruminal administration of ABZ at 10 mg kg-1(pasture group). After a three-week wash-out period, the same animals were housed and fed on a grain-based concentrate diet for three weeks prior to receiving the same ABZ treatment (concentrate group). Jugular blood and abomasal fluid samples were collected over 120 hours post-treatment. Plasma and abomasal fluid samples were analysed by high performance liquid chromatography (HPLC). The digesta transit time was measured using cobalt (Co) as a fluid marker; abomasal fluid and faecal samples were collected and Co concentrations measured by atomic absorption spectrophotometry. Complementary studies of the in vitro dissolution of ABZ particles at different pH values were also conducted. The pH of abomasal fluid collected from animals kept under both feeding conditions was registered. Increased concentrations of ABZ sulphoxide (ABZSO) and sulphone (ABZSO2) in plasma, resulting in significantly higher Cmax and area under the curve (AUC) values for both metabolites, were obtained in calves fed on the concentrate diet compared to those grazing on pasture. Enhanced abomasal fluid levels of ABZ and ABZSO were observed in concentrate-fed calves. The mean retention time of the digestive fluid marker in the gastrointestinal (GI) tract was significantly longer in the animals fed the grain-based diet. The in vitro dissolution of ABZ at a pH value equivalent to that obtained in the abomasum of the concentrate-fed calves (1.75) was significantly greater than that obtained at the pH registered in pasture-fed animals (2.00). The characterisation of the kinetic/metabolic behaviours and the resultant efficacy of antiparasitic drugs in animals reared under different management conditions may be relevant in increasing parasite control in livestock.     

In vitro inhibition of Eimeria tenella invasion by indigenous chicken Lactobacillus species

The aim of this study was to determine the effects of indigenous chicken Lactobacillus species isolates from different parts of the gastrointestinal tract on Eimeria tenella invasion in vitro and to characterise the nature of inhibition, if any. The effects of competitive exclusion, steric interference and bacterial extracellular factors on E. tenella invasion were examined in an MDBK cell model. Several Lactobacillus species were initially isolated from chickens and identified by biochemical characteristics and 16S-rRNA. All Lactobacillus species isolates tested, significantly inhibited E. tenella invasion. Steric interference did not affect parasite invasion. Extracellular metabolic factors secreted by Lactobacillus species isolates into the surrounding media were shown to inhibit parasite invasion and these factors appeared to be heat stable. These results show that the natural microflora of poultry can provide a source of E. tenella-inhibiting Lactobacillus species in vitro, and thus may contribute to the control of Eimeria infection.     

鸡艾美耳球虫18SrDNA序列与系统进化分析

为了确定鸡艾美耳球虫（Eimeria）不同种以及来自不同地区同种不同株之间的亲缘关系,研究其分类地位,对实验室保藏的柔嫩艾美耳球虫（Etenella）、毒害艾美耳球虫（Eneeatrix）、巨型艾美耳球虫（Emaxima）、堆形艾美耳球虫（Eaaervulina）等4种15株鸡球虫孢子化卵囊的18SrDNA基因进行克隆、测序,并与从GenBank下载的鸡球虫18SrDNA序列一起,使用软件DNAstar 5．0 MegAlign进行系统发育分析。结果显示,4种艾美耳球虫种间同源性在94．6％～99．4％之间,7株柔嫩艾美耳球虫的株间同源性在99．0％-99．9％之间,5株巨型艾美耳球虫的株间同源性在96．9％～99．8％之间。用该4种鸡球虫的18SrDNA序列与GenBank下载的另外4种鸡球虫18SrDNA序列构建系统发育树,显示这8种鸡艾美耳球虫形成2个分支,即堆形艾美耳球虫（EASH）、巨型艾美耳球虫（EMSH）、变位艾美耳球虫（Emivati）、和缓艾美耳球虫（Emitis）、布氏艾美耳球虫（Ebrunetti）、早熟艾美耳球虫（Epraecox）构成1个分支,柔嫩艾美耳球虫（ENSH）、毒害艾美耳球虫（ETAS）构成另1分支。巨型艾美耳球虫、柔嫩艾美耳球虫各株的系统发育树均根据地域关系产生2个分支。柔嫩艾美耳球虫、毒害艾美耳球虫的亲缘关系较近,不同地理区域的同种不同株的亲缘关系相对较远,种间和种内的鉴定结果与普通生物学结果一致。本研究提示18SrDNA基因可用于鸡球虫不同种／株的分类鉴定,为艾美耳球虫分子遗传学鉴定提供了理论基础。     

Comparative kinetic disposition of oxfendazole in sheep and goats before and during infection with Haemonchus contortus and Trich ostrongylus colubriformis

The kinetic disposition of [¹⁴C]-oxfendaEole (OFZ) and its metabolites, fenben-dazole (FBZ) and fenbendazole sulphone (FBZ.SO₂), in plasma and abomasal fluid were determined in Merino sheep and Angora goats before and during infection with Trichostrongylus colubriformis and Haemonchus contortus. The systemic availability (area under the plasma curve, AUC) of OFZ was significantly lower in goats (13.5 μg.h/ml) than in sheep (22.2 μg.h/ml) and was reduced with infection in goats (5.6 μg.h/ml) and sheep (15.1 μg.h/ml). The elimination of plasma [^l4C] was faster in goats than in sheep. The responses observed for [¹⁴C] were a reflection of the behaviour of OFZ. The concentration of OFZ and metabolites in abomasal fluid were similar in both species in the absence or presence of infection. However, as the mean flow rate of abomasal fluid was slower in goats (240 ml/h) than in sheep (488 ml/h), only 7% of the dose passed the pylorus in abomasal fluid of goats compared with 14% in sheep. The presence of gastrointestinal nematodes generally increased abomasal fluid flow rate but neither species nor infection had any effect on the rate or extent of [¹⁴C] excretion in urine or faeces. It is suggested that goats possess a faster hepatic metabolism than sheep resulting in more rapid elimination of OFZ.     

Viability of Sarcocystis neurona sporocysts after long-term storage

The effect of long-term storage on the viability and infectivity of Sarcocystis neurona sporocysts was investigated. S. neurona sporocysts were harvested from the small intestine of Virginia opossums from 1996 to 2002 and stored at 4 degrees C. Viability of sporocysts was assessed by propidium iodide (PI) exclusion assay, in vitro excystation and development in tissue cultures, and bioassay in gamma-interferon gene knockout (gamma-IFN-KO) mice. The rate of excystation was apparently unaffected by long-term storage; sporocysts retained their ability to excyst after 7 years of storage at 4 degrees C. However, the ability of sporocysts to exclude PI stain, to invade and proliferate in cells in vitro, and to cause disease and lesions in gamma-IFN-KO mice appeared to decline as sporocysts age. The results demonstrated that sporocysts of S. neurona were able to survive and maintain moderate to high viability for up to 7 years when stored in phosphate buffered saline and Hank's balanced salt solution containing antibiotic-antimycotic mixture at 4 degrees C.     

Distribution of oocysts, sporocysts and sporozoites of Eimeria tenella and Eimeria maxima in the digestive tract of chicken.

The distribution of oocysts, sporocysts and sporozoites of Eimeria tenella and Eimeria maxima in the digestive tract of chicken and in excreta was investigated. At 1 h after the oral inoculation of E. tenella oocysts, the number of sporocysts in the cecum was 3.4 x 10(6) and decreased gradually thereafter, and the number of sporozoites in the cecum increased and remained at a high level until 12 h after the inoculation. Small numbers of sporocysts and sporozoites of E. tenella were found in other intestinal sites. A great number of E. maxima sporozoites was found, especially in the jejunum, 2 h after the inoculation. The findings that the largest populations of sporozoites of E. tenella and E. maxima were found in the cecum and the jejunum, respectively, indicate that the site specificity of sporozoite invasion for each species is determined before the invasion takes place.     

Use of monoclonal antibodies directed against Eimeria tenella sporozoites to determine stage specificity and in vitro effect on parasite penetration and development

Three different hybridoma-produced monoclonal antibodies (Ab) were used to study their reactivities with in vitro developmental stages of Eimeria tenella and their effects on sporozoite penetration and intracellular development. One Ab (designated B10) was stage-specific, whereas the other 2 Ab (designated C3 and E5) reacted with various intracellular developmental stages of the coccidia. The E5 Ab interacted with the cytoplasm of cultured cells that were infected with sporozoites at 24 hours after inoculation. All 3 Ab inhibited penetration to various degrees--the one designated B10 having the greatest inhibitory effect. These 3 Ab also inhibited development of the parasite in cell culture, provided that Ab was continuously present in the cell culture medium. Removal of Ab from the medium allowed coccidial development to continue at about the same rate as in controls. A longer pretreatment time of the sporozoites with the Ab before cell inoculation increased the inhibitory effect with respect to both the penetration and the development of the parasite.     

Anticoccidial evaluation of halofuginone, lasalocid, maduramicin, monensin and salinomycin

The activities of five anticoccidials were compared against Eimeria species in/of chickens, in controlled in vivo and in vitro laboratory studies. Two more recent and potent market entries (maduramicin and halofuginone) were compared with three older polyether antibiotic anticoccidials (monensin, lasalocid and salinomycin). Halofuginone, lasalocid, maduramicin, monensin and salinomycin were evaluated at 3, 125, 5, 120 and 66 ppm, respectively, of active drug in the diets. At these levels, all five drugs demonstrated significant activity against Eimeria tenella, E. maxima, E. necatrix, E. brunetti and E. acervulina (in vivo). Monensin was least effective against E. tenella, and one of the lesser efficacious drugs against E. necatrix, maduramicin, was least effective against E. maxima. In studies of single Eimeria species infections, comparable weight gains were noted for the drugs. In the mixed Eimeria species infections, however, birds treated with maduramicin had significantly higher weight gains than did birds medicated with monensin. Unlike in vivo potencies, titration in vitro indicated that monensin was most potent (active at 10(-6) mcg ml-1), and maduramicin and lasalocid least potent (inactive at less than or equal to 10(-3) mcg ml-1).     

Evaluation of interspecific hybrids of the chicken, guinea fowl, and Japanese quail for innate resistance to coccidia

Experimental chicken/guinea fowl hybrids, guinea fowl, and chickens were orally inoculated with Eimeria acervulina or E. tenella, which are specific for chickens, or with E. grenieri, which is specific for guinea fowl. No intact oocysts were found in feces within 24 hr of inoculation, suggesting that excystation occurred in the normal and abnormal hosts. No oocysts were found in the feces of hybrids during a 9-day postinoculation period. The guinea fowl passed oocysts of guinea fowl coccidia (E. grenieri) but not those of chicken coccidia, and the chickens passed oocysts of chicken coccidia (E. acervulina and E. tenella) but not those of guinea fowl coccidia. Some asexual development (schizogony) occurred in hybrids inoculated with E. tenella, but sexual development (gametogony) did not. In contrast, quail/chicken hybrids became infected with oocysts of chicken coccidia (E. acervulina, E. tenella, and E. maxima) and quail coccidia (E. bateri) and passed a few oocysts during the normal patent period; control chickens and quails became heavily infected with oocysts of chicken and quail coccidia, respectively.